Our understanding of gene expression regulation has changed radically with the discovery of ribonucleoprotein (RNP) complexes programmed with small RNAs. At the core of these complexes are members of the Argonaute family of proteins that is comprised of two subclades: Ago and Piwi. Ago proteins bind to microRNAs. Piwi proteins bind to piwi-interacting RNAs (piRNAs). Piwi proteins are essential for germline development and germ cell specification. However, the function and RNA targets for many piRNAs and Piwi proteins are largely unknown. Genetic studies in Drosophila melanogaster have identified maternal genes whose products assemble in structures known as germ plasm, and which are required for germ cell specification. Among these factors are the piRNA binding protein Aubergine, an arginine protein methyltransferase (PRMT5) and its cofactor, the Tudor protein and the Vasa RNA helicase. Although the genetic pathway of germ plasm assembly has been well characterized in Drosophila, the relationship and functions of germ plasm proteins is not well understood. Germ plasm also contains RNAs, that are thought to be essential for germ cell specification but whose identities are mostly unknown. Most of the genes that are required for germline development and germ cell specification in Drosophila have evolutionary conserved roles in the germline of other animals, such as mice. Our studies are uncovering important relationships and functions of critical germ plasm components. We find that arginine methylation of Piwi proteins, catalyzed by PRMT5, is an evolutionary conserved modification that mediates interaction with Tudor proteins. We also find that Vasa and its mouse homolog, contain methylated arginines. In Aim 1, we will functionally characterize piRNP complexes and their RNA targets in Drosophila melanogaster. In Aim 2, we will functionally characterize piRNP complexes and their RNA targets in Mus musculus. Studying both organisms will highlight conserved roles and pathways for piRNP function and germ cell specification and development, but will also likely uncover species-specific functions. The studies detailed in this proposal will address fundamental aspects of the function of piRNPs and associated proteins and their RNA targets, and the role of these factors in germ cell specification and germline development.